Novel α-N-heterocyclic thiosemicarbazone complexes: synthesis, characterization, and antimicrobial of properties investigation

In this paper, eight novel α-N-heterocyclic thiosemicarbazone complexes were synthesized in search of new biologically active compounds, and characterized via organic elemental analysis, nuclear magnetic resonance spectroscopy, infrared spectra, thermogravimetric analysis, ultraviolet-visible spectroscopy, molar conductance and magnetic susceptibility measurements. The in vitro antimicrobial activity of these complexes was examined against ten disease-causing pathogens: Gram-positive bacteria (Micrococcus luteus ATCC9341, Staphylococcus epidermidis ATCC12228, Bacillus cereus RSKK863) and Gram-negative bacteria (Pseudomonas aeroginosa ATCC27853, Klebsiella pneumonia ATCC27853, Enterobacter aerogenes ATCC51342, Salmonella typhi H NCTC9018394, Shigella dysenteria NCTC2966, Proteus vulgaris RSKK96026) and yeast (Candida albicans Y-1200-NIH). The results revealed that the α-N-heterocyclic thiosemicarbazone compounds showed potent activity. It was observed that all thiosemicarbazone complexes were more susceptible to Gram-negative strains based on the presence of an electron-withdrawing substituent (–Br/–Cl/–F). It was determined that thiosemicarbazone Cu2+complexes showed stronger antifungal effects.


Introduction
Heterocyclic compounds are organic structures in which some carbon atoms are replaced by heteroatoms such as nitrogen, sulphur, oxygen, and phosphorus. 1 Heterocyclics have different physical and chemical properties and reactivity depending on the heteroatoms' ring size and structure. 2Recently, there has been an increasing interest in heterocyclic compounds due to their various applications.The synthesis of innovative, stereoselective, functional new heterocyclic compounds is of great interest for drug discovery and development. 3Heterocyclic compounds are used in the agrochemical industry in crop protection due to their pesticidal activities. 4Heterocycles are used in the pharmaceutical industry to treat some diseases (such as Alzheimer's, cancer, diabetes, circulatory diseases, and AIDS) due to their therapeutic properties. 5Heterocycles bind with enzymes due to their variety of intermolecular interaction properties.These properties of heterocycles are the reasons for preference in anti-cancer drug design. 6Particularly, thiosemicarbazone-based heterocyclics are an important class of heterocyclic compounds because of their coordination capacity. 7They are important chelating ligands due to the containing potential donor atoms (deprotonated phenolic oxygen, thione/thiol sulphur, azomethine nitrogen, etc.). 8specially, a-N-heterocyclic thiosemicarbazones, in which the thiosemicarbazone side chain is linked to an N-heterocyclic ring at the a position, are strong metal chelating agents. 9Heterocyclic compounds containing thiosemicarbazone have various biological, cytotoxic, and pharmacological activities.These properties of thiosemicarbazones are generally related to the presence of imine group (-N]CH-) and metal ion coordination.The coordination affects such properties as lipophilicity, drug resistance, etc. [10][11][12] The complexes can exhibit bioactivities not shown by the free ligands. 134][25][26] Thiosemicarbazone transition metal complexes have been reported as extensively effective and preferred drugs. 27,28The synthesis of new heterocyclic thiosemicarbazone metal complexes is important due to their biological and pharmacological properties.Therefore, novel a-N-heterocyclic thiosemicarbazone complexes were synthesized using thiosemicarbazide, carboxaldehyde, aldehyde derivatives, and metal salts.
It is predicted that the new heterocyclic thiosemicarbazone Ni 2+ and Cu 2+ complexes synthesized within the scope of this study will contribute to the importance of heterocyclic chemistry in the pharmaceutical industry and medicinal chemistry.

Materials and measurements
All chemicals were procured from Sigma-Aldrich.Organic elemental analyses were obtained with a Thermo-Scientic Flash-2000 model elemental analyzer.IR spectra were recorded from KBr pellets using a Shimadzu IR Prestige-21 model spectrometer. 1H-NMR spectra were measured on a Bruker Biospin brand Avance III 400 MHz model device.UV-Vis spectra were determined on a UV-1800 ENG240V, So model spectrophotometer.TGA analysis were carried out using a Shimadzu DTG 60H-DSC 60 model thermal analyzer.The molar conductance of complexes was measured in dimethyl sulphoxide (DMSO) at 21 °C on Conductivity 430 Lab.Magnetic measurements were obtained with a Sherwood Scientic MKI model Evans magnetic susceptibility device.

Synthesis of heterocyclic compounds containing thiosemicarbazone
All heterocyclic thiosemicarbazone Ni 2+ and Cu 2+ complexes were prepared with the template method (Fig. 1 and 2).Novel heterocyclic thiosemicarbazone complexes (H 1 Ni, H 2 Ni, H 3 Ni, H 4 Ni, H 1 Cu, H 2 Cu, H 3 Cu, and H 4 Cu) were synthesized by the reaction of 4-phenyl-thiosemicarbazide (4 mmol) and 2aminothiazole-5-carboxaldehyde (4 mmol) in ethanol/DMSO mixture.The pH of the solution was adjusted to 5-5.5 with 1 mL of acetic acid.The solution was heated with stirring under reux for 5 h at 80 °C.The ethanol solution (50 mL) of the salicylaldehyde derivatives (4 mmol) was added to the mixture and stirred for a further 5 h at 80 °C.5-Fluoro-3methylsalicylaldehyde, 5-bromo-salicylaldehyde, 3-chloro-5-uorosalicylaldehyde, and 5-methylsalicylaldehyde were used as salicylaldehyde.The ethanol solution (5 mL) of the metal salts [nickel(II) chloride hexahydrate & copper(II) chloride (anhydrous)] was added dropwise to the reaction mixture and stirred by reuxing for a further 4 h at 70 °C.The mixture was evaporated at room temperature.The colored product was ltered, puried, and dried.

Antimicrobial assay
Gram (+) bacteria, Gram (-) bacteria and yeast used in the antimicrobial study are as follows, respectively: (Micrococcus luteus ATCC9341, Staphylococcus epidermidis ATCC12228, Bacillus cereus RSKK863), (Pseudomonas aeroginosa ATCC27853, Klebsiella pneumonia ATCC27853, Enterobacter aerogenes ATCC51342, Salmonella typhi H NCTC9018394, Shigella dysenteria NCTC2966, Proteus vulgaris RSKK96026) and (Candida albicans Y-1200-NIH).0][31] In this method, it was determined that DMSO, used as solvent control, did not show antimicrobial activity against the tested organisms.As a rst step, all heterocyclic thiosemicarbazone complexes were solved (3.5 mg mL −1 ) in DMSO, and all pathogenic microorganisms were incubated in Nutrient Broth agar (10 6 CFU mL −1 ) for 24 h at 37 °C.As a second step, these cultures were then homogenized by adding them to Mueller-Hinton Agar (MHA) cooled to 45 °C, and they were poured into sterile Petri dishes and cooled.Aerward, wells of 6 mm diameter were pierced in these agars, and the heterocyclic thiosemicarbazone complexes were added.Finally, the plates were incubated in an oven (at 37 °C, 24 h), the inhibition zone of all heterocyclic compounds was measured, and then the average of the activity values performed with two repetitions was taken.As a third second, selected disease agent pathogens were compared with standard antibiotics.For this purpose, Ampicillin (AMP10), Sulphamethoxazole (SXT25), Amoxicillin (AMC30), and Kanamycin (K30) antibiotics were used for Gram (+) and Gram (-) bacteria, and Nystatin (NYS100) antibiotic was used for yeast.

Characterization of heterocyclic compounds containing thiosemicarbazone
Some physical properties, analytical, and organic elemental analysis data of all heterocyclic thiosemicarbazone Ni 2+ and Cu 2+ complexes are presented in Table 1.It was dened that the elemental analyses and the chemical formulas of all heterocyclic thiosemicarbazone complexes were compatible.
IR spectra data of all heterocyclic thiosemicarbazone Ni 2+ and Cu 2+ complexes are presented in Table 2 and are shown in Fig. S1.† In the infrared spectra of all complexes, absorption bands were observed in 1003-1017 cm −1 and 3219-3247 cm −1 ranges, which were assigned to the n(N-N) and n(N-H) vibrations, respectively.The stretching vibrations of n(CH]N) belonging to the azomethine groups obtained by the condensation reaction of aldehydes and amines, were observed in the 1603-1621 cm −1 and 1501-1544 cm −1 ranges, respectively.The absorption bands assigned to n(C]C) and n(CH) of the aromatic ring were observed in the 1453-1495 cm −1 and 3025-3080 cm −1 ranges, respectively.The stretching vibrations of n(C-S-C) belonging to the thiazole groups were determined in the 738-753 cm −1 region.The stretching vibrations of n(C]S) vibrations occurred in the 815-842 cm −1 and 1203-1212 cm −1 ranges, respectively.Additionally, the observation of bands in the ranges 479-504 cm −1 and 559-596 cm −1 was due to the stretching vibrations of n(M-N) and n(M-O). 32These bands indicate the coordination of ligands to metal centers.
1 H-NMR spectra data of all heterocyclic thiosemicarbazone Ni 2+ complexes are presented in Table 3   UV-Vis spectra data of all heterocyclic thiosemicarbazone Ni 2+ and Cu 2+ complexes are presented in Table 4.The electronic spectra of all nickel and copper complexes showed two main bands.p / p* transitions of the aromatic ring and n / p* transitions of the imine group were observed in the ranges of 231-252 nm and 323-327 nm, respectively.In the electronic spectra of nickel complexes, the absorption bands that appeared in the 377-383 nm range were assigned to metalligand charge transfer transitions.The bands observed in the 659-663 nm range were assigned to the d-d transitions, which is compatible with the Ni 2+ square planar geometry. 34In the electronic spectra of copper complexes, the absorption bands observed in the 428-441 nm range were assigned to intra-ligand charge transfer transitions. 35The bands that appeared in the 887-893 nm range were assigned to the d-d transitions, which is compatible with the Cu(II) tetrahedral geometry. 36e magnetic moments of heterocyclic thiosemicarbazone Ni 2+ complexes were determined diamagnetic, conrming the existence of square planar geometry. 37The magnetic moments of heterocyclic thiosemicarbazone Cu 2+ complexes were observed in the range of 1.81-2.11B.M., indicating the presence   of one unpaired electron in the Cu 2+ ion. 38The paramagnetic behavior conrms the existence of tetrahedral geometry. 39he molar conductance of heterocyclic thiosemicarbazone Ni 2+ and Cu 2+ complexes was determined in the range 2.5-35.6 mS cm −1 and 15.4-55.7 mS cm −1 , respectively, conrming the non-electrolyte nature of all complexes in DMSO solution. 40e graphical illustration and the photographs of inhibition regions of pathogenic bacterial species are presented in Fig. 3-5, respectively.The heterocyclic thiosemicarbazone Ni 2+ and Cu 2+ complexes' were tested in vitro against selected diseasecausing pathogenic bacteria and yeast to determine their antibacterial and antifungal activities.The yeast and pathogens  In conclusion, the heterocyclic thiosemicarbazone Ni 2+ and Cu 2+ complexes were determined to have high or moderate antifungal and antibacterial activity.It was observed that the antibacterial and antifungal activities of thiosemicarbazone compounds, including electron-withdrawing groups (-Br/-Cl/-F), were more effective than the thiosemicarbazone compounds, including electron-donating group (-CH 3 ).The synthesized thiosemicarbazone compounds showed very good antibacterial and antifungal properties because they contain thiazole rings containing N and S heteroatoms, nickel and copper metal ions with chelation ability, and asymmetric diimine groups that impart biological activity.This situation is consistent with similar research results. 24,48,49The antimicrobial activities vary depending on the presence of metal ions, the position of the substituent on the ring, and the presence of terminal groups (-CH 3 , -Ph, -F, -Cl, -Br, etc.).Increased and/or decreased biological activities have been reported for compounds. 27The antimicrobial activities increase and/or decrease depending on the presence of metal ions, the position of the substituent on the ring, and the presence of terminal groups (-CH 3 , -Ph, -F, -Cl, -Br, etc.).Ni 2+ complexes generally showed stronger antibacterial activity than Cu 2+ complexes.Especially the H 2 Ni complex exhibited a higher inhibitory effect.

Conclusions
In this study, novel a-N-heterocyclic thiosemicarbazone compounds were synthesized using the template method and were characterized by different spectroscopic techniques.Based on the spectral studies, it was determined that Ni 2+ complexes had a square planar geometry, while Cu 2+ complexes had tetrahedral geometry.The biological activities of heterocyclic thiosemicarbazone compounds were evaluated against diseasecausing pathogens using the well-diffusion method.All heterocyclic thiosemicarbazone complexes were observed to have high or moderate antimicrobial activity.The synthesized thiosemicarbazone compounds containing asymmetric azomethine groups exhibited different biological activities depending on the presence of metal ions and the presence of terminal groups.The heterocyclic thiosemicarbazone Ni 2+ and Cu 2+ complexes exhibited more potent impacts against different Gram-negative bacterial strains as potential antibacterial agents.The heterocyclic thiosemicarbazone Ni 2+ complexes showed more potent impacts against yeast as potential antifungal agents.According to high/or moderate biological activity results, the synthesized novel a-N-heterocyclic thiosemicarbazone compounds can be recommended as potent inhibitors in diverse pharmaceutical, biological, medicinal, biomedical, etc. applications.

Table 1
Some physical properties, analytical and organic elemental analysis data of heterocyclic thiosemicarbazone complexes a a D: diamagnetic.

Table 2
FT-IR spectra data of heterocyclic thiosemicarbazone complexes

Table 4
TGA and UV-Vis data of heterocyclic thiosemicarbazone complexes